Many DNA tumor viruses encode "initiators" proteins that site specifically bind to viral origins of DNA replication. Upon binding to their respective origins, the initiators assemble into DNA helicases that are capable of melting duplex DNA. Initiators also recruit cellular proteins required for synthesis of nascent DNA. We are attempting to understand these processes by characterizing in depth an initiator encoded by Simian Virus 40, termed T-antigen (T-ag). Using the Simian Virus 40 system and simple band shift experiments, a fundamental question in biology will be addressed: "how does the cell cycle machinery control the assembly of initiators on viral origins of replication?" Recent experiments with T-ag derived peptides, whose ability to bind to DNA is regulated by phosphorylation of Thr 124, are providing significant insights into this process. Experiments are proposed to determine if similar mechanisms are operating in the context of T-ag. Related aspects of T-ag assembly and regulation will be considered, such as locating a site on T-ag whose interactions with the flanking sequences in the core origin is necessary for T-ag binding. Collectively, these studies will provide information that may allow us to solve the mechanism of DNA unwinding. Once the SV40 origin is unwound, the pol alpha-primase complex initiates the synthesis of nascent DNA strands. Exactly what sequences constitute the initiation sites for the pol alpha-primase complex is the topic of an additional series of experiments. Given that many basic processes are conserved throughout evolution, we are confident that the information we obtain from these studies will be pertinent to the initiation of DNA replication at other viral origins of replication. Furthermore, they may help us to understand how the cell cycle machinery controls initiation events at cellular origins. Given that uncontrolled DNA replication is thought to be a component of many diseases, including cancer, it is very important to understand how DNA replication is initiated and how it is controlled.